Engineering and functionalization of large circular tandem repeat protein nanoparticles

Correnti, Colin; Hallinan, J.P.; Doyle, Lindsey; Ruff, Raymond O.; Jaeger-Ruckstuhl, Carla A.; Xu, Yuexin; Shen, Betty; Qu, Amanda; Polkinghorn, Caley; Friend, Della; Bandaranayake, Ashok D.; Riddell, Stanley R.; Kaiser, Brett K.; Stoddard, Barry; Bradley, Philip

doi:10.1038/s41594-020-0397-5

Cited by 29 publications

(44 citation statements)

References 21 publications

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“…Unlike human antibodies, antigen-binding domains (VHHs) derived from camelid single-chain antibodies are ideal for expression in prokaryotes, like spirulina, because neither intracellular formation of disulfide bonds nor specific glycosylation is required for synthesis of the bioactive protein 29 . VHHs were constitutively expressed in spirulina from the strong promoter Pcpc600 in various formats, including monomers, dimers 30 , trimers 31 and heptamers 32 (Supplemental Figure S2). Monomeric VHHs were typically expressed as a fusion protein with a solubilityenhancing chaperone, such as the E. coli maltose-binding protein (MBP).…”

Section: Expression Of Camelid Single-chain Antibody Fragments In Spimentioning

confidence: 99%

Expression and manufacturing of protein therapeutics in spirulina

Jester¹,

Zhao²,

Gewe³

et al. 2021

Preprint

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Arthrospira platensis (commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina’s genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

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Section: Expression Of Camelid Single-chain Antibody Fragments In Spimentioning

confidence: 99%

Expression and manufacturing of protein therapeutics in spirulina

Jester¹,

Zhao²,

Gewe³

et al. 2021

Preprint

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“…To ensure rigid structurally coherent junctions between the building blocks, we only allow fusion via alignment and superposition of shared helices from both building blocks being fused and disallow α-helical extension. Most globular proteins are destabilized by truncation in the midst of secondary structure elements; to maintain stability, we use idealized repeat protein building block spacers (and oligomers, when possible), where every repeat unit is identical – such proteins are amenable to truncation or fragmentation without undermining folding and stability 27-30 .…”

Section: Resultsmentioning

confidence: 99%

Generation of ordered protein assemblies using rigid three-body fusion

Vulovic

Yao

Park

et al. 2020

Preprint

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Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

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“…protein complexes are attractive building blocks for making higher order 2D arrays and 3D crystal protein assemblies, and can be useful for receptor clustering in cellular engineering 31 . We first set out to design dihedral protein assemblies of D2 symmetry.…”

Section: Generation Of Two-component Dihedral Assemblies : Dihedral Smentioning

confidence: 99%

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

Hsia¹,

Mout²,

Sheffler³

et al. 2020

Preprint

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A goal of de novo protein design is to develop a systematic and robust approach to generating complex nanomaterials from stable building blocks. Due to their structural regularity and simplicity, a wide range of monomeric repeat proteins and oligomeric helical bundle structures have been designed and characterized. Here we describe a stepwise hierarchical approach to building up multi-component symmetric protein assemblies using these structures. We first connect designed helical repeat proteins (DHRs) to designed helical bundle proteins (HBs) to generate a large library of heterodimeric and homooligomeric building blocks; the latter have cyclic symmetries ranging from C2 to C6. All of the building blocks have repeat proteins with accessible termini, which we take advantage of in a second round of architecture guided rigid helical fusion (WORMS) to generate larger symmetric assemblies including C3 and C5 cyclic and D2 dihedral rings, a tetrahedral cage, and a 120 subunit icosahedral cage. Characterization of the structures by small angle x-ray scattering, x-ray crystallography, and cryo-electron microscopy demonstrates that the hierarchical design approach can accurately and robustly generate a wide range of macromolecular assemblies; with a diameter of 43nm, the icosahedral nanocage is the largest structurally validated designed cage to date. The computational methods and building block sets described here provide a very general route to new de novo designed symmetric protein nanomaterials.

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Engineering and functionalization of large circular tandem repeat protein nanoparticles

Cited by 29 publications

References 21 publications

Expression and manufacturing of protein therapeutics in spirulina

Expression and manufacturing of protein therapeutics in spirulina

Generation of ordered protein assemblies using rigid three-body fusion

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

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